High-Level Secretory Production of Recombinant E2-Spy Antigen Protein via Combined Strategy in Pichia pastoris

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Nothing specific to mention except specify line 222 to what the comparison refers to.

Author Response

Reviewer 1 recommended modifying the sentence on line 222 for clarity. The revised sentence reads, "To facilitate comparison, the mRNA levels and ES expression levels were comparatively presented using relative quantification methods (normalized to the control strain 1-α-ES, Lane 1)." Likewise, the figure legends for Figures 3 to 9 were also adjusted.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Although some mRNA analysis data have been obtained and included into the manuscript, I cannot admit that the manuscript was sufficiently improved.

1. The mRNA analysis lacks proper controls. The RNA preparations sometimes are contaminated with genomic DNA and the PCR products can be amplified from the genomic DNA instead of cDNA. That is why the strain with the empty vector is not a proper control. The RNA isolated from the cells grown in promoter repression conditions and samples before reverse transcription could serve as negative controls. Besides, there is no data on mRNA levels in experiments with molecular chaperon co-expression (Figures 8 and 9).

2. I do not understand why the Western blot analysis of intracellular ES was not included. This could provide valuable data on the ES expression level and its folding efficiency in the yeast secretory pathway. Misfolded protein can accumulate within the ER as a core-glycosylated form producing distinct band on SDS-PAGE (for example see https://link.springer.com/article/10.1007/BF00427036  or  https://link.springer.com/article/10.1186/1471-2199-3-15 ).

3. The writing style has not been improved. There are too many issues through the text and it is difficult to list all of them. The Discussion looks like a mini review describing post-translational translocation, which is not directly related to the obtained results. There is no any data showing whether the secretion leaders used direct co- or post-translational translocation of ES.

 

Minor points:

Line 45 - The statement “pro-region provides relevant information for the transport from ER to Golgi” is misleading.

line 46 - The statement “The α-factor signal peptide guides precursor proteins into the ER through the post-translational translocation pathway [7]” is also misleading. Reference 7 describes in vitro translocation of pre-pro-alpha-factor in yeast S. cerevisiae. This does not mean that any protein provided with alpha-factor pre-pro leader would be post-translationally translocated in vivo in any yeast species.

line 66 – “was increased by 17.87-fold (as determined by Western blot)” – I am impressed by preciseness of Western blot analysis (also see lines 18, 22, 24 and so on).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

In the revised version the authors have improved the manuscript significantly. I have no further comments.

Author Response

We are grateful for your acknowledgment of this manuscript.

Round 2

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

None of my concerns have been addressed. The presence the DNA removal reagents in the RNA analysis kit does not mean that DNA has been removed. This should be proved. Some “corrections” introduced into the text either did not improve or made the situation even worse. For example, lines 43-46: terms secretion signal and pre-sequence are synonymous, while pro-region is not a part of secretion signal; the statement “pro-region mediates receptor-dependent packaging into ER-derived COPII transport vesicles” might be true in case of S. cerevisiae alpha-factor (though I don’t know about studies directly showing this), but it is wrong for many other proteins having pro-regions.

I cannot recommend this manuscript for publication before additional analyses are performed (controls for RT-PCR, Western blotting of intracellular protein). Complete re-writing of Discussion is also absolutely required: reviewing literature, which is not directly related to the experimental data, should be avoided.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Li et al have worked on developing a strategy to express high-levels of recombinant E2-Spy protein antigen. They have investigated the use of different signal peptides and varied copy number to see its effect on protein expression. The authors also worked on employing co-expression of molecular chaperones to assist in the increased production of E2-Spy protein. The authors have covered all the concerns raised in the previous round of reviews so I am happy to recommend this manuscript for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The main problems of the manuscript indicated in my previous review have not been solved. As I mentioned before, proper conclusions can be drawn from the data presented in the manuscript only after assaying mRNA levels e.g. by the Northern blot analysis. The writing style has not been improved. The discussion section has become even worse after revision. I have many concerns regarding the language quality, however I don’t think it is worthy of listing all of them, since it would not solve the main problems of the manuscript.

Author Response

We greatly appreciate your valuable feedback, which is essential for ensuring the manuscript's overall quality, a common objective for all involved. In our subsequent revisions, several improvements were made.

Firstly, qPCR and 2^-ΔΔCT method were utilized to detect the transcription levels of ES in cases where variations in the signal peptide or ES gene dosage occurred (Figure 2 to Figure 6). The evaluation of ES transcription levels was not undertaken for Figure 7 to Figure 10 as there were no changes in the signal peptide or gene dosage of ES. Furthermore, attempts were made to detect the intracellular accumulation of ES using Western blot assays. The results revealed that the glycosylation modification of ES led to smeared protein bands, thus obscuring differentiation between the various strains. Secondly, figures and figure legends were revised using numbers rather than sample names. Lastly, the introduction and discussion sections underwent extensive rewrites. The introduction briefly outlined the background of signal peptides, gene dosage, and molecular chaperones, while the discussion focused on providing potential explanations for the observed phenomena and delineated future research directions for improvements. All modifications have been highlighted in the revised manuscript for clarity and comparison. The revisions were so numerous that not every specific line number is listed here. 

Please refer to the attachment for more information.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed many of the remarks made previously. I would like to point out that although genome integration has been the only way to modify the Komagataella pastoris genome, since 2018 it is possible to transform it with stable centromeric plasmids (Nakamura 2018 in Appl Envriron Microbiol).

The only information missing in the materials and methods is the way proteins are harvested from the culture medium, TCA or ammonium sulfate precipitation. Do the authors use another method or directly load the culture medium?

One issue the authors do not discuss is the fact that the AOX1 promoter drives the expression of a protein that undergoes cytoplasm to peroxisome transport and use it to express a protein that will undergo a cytoplasm to ER transport. To what extend does the 5'UTR influence the efficiency of translation at ER ribosomes? Are inducible promoters driving the expression of plasma membrane proteins used in Komagataella pastoris. Have the authors envisaged using the PMA1 promoter?

Figure 4 is probably best introduced in the supplementary data and made bigger for easy of read.

The discussion is too long and elements of the first 4 paragraphs are better placed in the introduction where the notions of proprotein signals and preprotein signals are not well explained.

Comments on the Quality of English Language

Nothing to say on the quality of the English. Noted a small typo line 223, space missing between full stop and Contrary.

Author Response

Please see the attachment.

Author Response File: Author Response.docx